X-RAY SPECTROSCOPIC STUDIES ON HEME-COPPER OXIDASES

Project: Research project

Description

DESCRIPTION: Heme-copper oxidases function to couple the energy of oxygen reduction to ATP synthesis in the process of respiration. Thus, these redox enzymes play pivotal roles in aerobic metabolism. The redox processes occur at the metal centers which constitute the active sites of the enzymes and are easily inhibited by small molecules such as cyanides, carbon monoxide and other toxic substances, often with fatal consequences. Understanding the coordination chemistry of the metal centers is a critical step in elucidating the detailed mechanism of respiration and energy transduction within the cell. The recent publication of crystal structures that define the coordination of the metal centers has settled a number of old controversies but has also raised new and even more intriguing questions that can only be addressed by well-focused spectroscopic experiments. This proposal seeks funding to apply X-ray absorption spectroscopy (XAS) to a number of important structural and mechanistic questions highlighted by the crystal structural data. The proposed studies utilize the unique sensitivity of XAS to refine further the metal ion coordination in the oxidized enzyme, and to probe for the first time the coordination present in the reduced enzyme and the catalytic intermediates P and F. The results of these experiments will be used to test the validity of the "histidine cycle,' a recently proposed mechanism for proton pumping. The proposed studies will also investigate in detail the structure of the CuA center in the soluble subunit II fragment, as well as in a number of engineered constructs prepared by "loop-directed" mutagenesis. These experiments follow on from the initial determination from EXAFS of a unique 2.34 A Cu-Cu interaction in CuA. Other experiments are planned that will explore the metal-site coordination in two novel heme-copper oxidases, nitric oxide reductase and azurin oxidase.
StatusFinished
Effective start/end date5/1/972/29/16

Funding

  • National Institutes of Health: $261,067.00
  • National Institutes of Health: $262,003.00
  • National Institutes of Health
  • National Institutes of Health: $261,391.00
  • National Institutes of Health: $218,695.00
  • National Institutes of Health: $280,772.00
  • National Institutes of Health: $218,864.00
  • National Institutes of Health: $216,342.00
  • National Institutes of Health
  • National Institutes of Health: $74,240.00
  • National Institutes of Health: $238,210.00
  • National Institutes of Health: $238,210.00
  • National Institutes of Health
  • National Institutes of Health: $159,100.00
  • National Institutes of Health: $252,543.00
  • National Institutes of Health: $238,210.00
  • National Institutes of Health: $240,960.00

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Heme
X rays
X ray absorption spectroscopy
Copper
Metals
Oxidoreductases
Electron Transport Complex IV
Histidine
Protons
Enzymes
Metallochaperones
Metal ions
Mutagenesis
Electrons
Proteins
copper oxidase
Crystal structure
Experiments
Bacilli
Azurin

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)